MAPK and pro-inflammatory mediators in the walls of brain blood vessels following cerebral ischemia

INTRODUCTION Stroke is a serious neurological disease which may lead to death and severe disability [1, 2]. There are two major types of stroke: ischemic and hemorrhagic stroke. Both are associated with disruption of blood flow to a part of the brain with rapid depletion of cellular energy and oxygen, resulting in ionic disturbances and eventually neuronal cell death [3]. The pathologic process that develops after stroke is divided into acute (within hours), sub-acute (hours to days), and chronic (days to months) phases [4, 5]. Obviously, the most effective therapy requires the earliest possible intervention e.g. with removal of a thrombus. However, no specific treatment, apart from thrombolysis, that acts effectively to protect the neurons during the acute phase has yet been developed. Experimental and clinical data show an acute and prolonged inflammatory response in the brain after a stroke. Several investigators have reported that inflammation evolves within a few hours after stroke, and plays an important role in the development of the cerebral lesions [6]. This inflammatory reaction involves activation of resident cells (mainly microglia), infiltration and accumulation of various inflammatory cells (including neutrophils, leukocytes, monocytes, macrophages), and production of pro-inflammatory mediators in the injured brain areas [6, 7]. It has been established that the inflammatory reaction triggered by stroke affects not only the neuronal tissue itself but has impact also on the cerebral arteries [7]. Stroke is a vascular disease and despite extensive research in the area, the physiology and pathophysiology of the neurovascular unit, the complex network of endothelial cells, smooth muscle cells, inflammatory cells and mediators are not fully understood, which is necessary in order to develop effective therapies. The aim of the present thesis was to examine the role of pro-inflammatory mediators in cerebrovascular pathophysiology following stroke. The main focus was directed towards the expression and production of cytokines and inducible nitric oxide synthase (iNOS), the activation of matrix metalloproteinases (MMPs) and mitogen activated protein kinase (MAPK) pathway because microarray work [8] and published data [9] primarily pointed at these. These parameters and the relationships between them were studied in the cerebrovascular walls after ischemic and hemorrhagic strokes. This study lends further support to the view that inflammatory mediators are important contributing factors in brain injury after stroke. It provides evidence that blocking the intracellular signaling pathways involved in the transcription of these mediators may have therapeutic potential, as it may prevent or at least attenuate the inflammatory processes elicited by stroke. Ischemic stroke Ischemic stroke is the most common type of stroke (85% of cases). It is caused by a transient or permanent occlusion of a cerebral artery most often by a thrombus or an embolus [10, 11]. When an ischemic stroke occurs, blood flow to an area of the brain is reduced and the brain cells are starved of oxygen and nutrients, which quickly leads to neuronal cell death and the development of an infarct. The infarct region is divided into two parts: 1) A central part or an ischemic core, where the neurons die and have no chance to survive without rapid reperfusion. 2) A peripheral area or an ischemic penumbra, which surrounds the core [12]. Cells in the penumbra are impaired and cannot function due to compromised metabolism, but do not die immediately and have therefore become a prime target for neuroprotective treatments [13-15]. A number of neurochemical and pathophysiological events are triggered within the ischemic penumbra. As a result of energy depletion, there is disruption of ion homoeostasis, excessive release of excitatory neurotransmitters such as glutamate, calcium channel dysfunction, generation of oxidative stress and free radicals, activation of stress signaling, cell membrane disruption, inflammation, ultimately leading to necrotic and apoptotic cell death [1, 4, 15, 16]. The effect of ischemia on brain cells results not only in loss of structural integrity of brain tissue but affects also blood vessels, partly through the activation of inflammatory events and excess production of vasoconstrictor substances and increased receptor expression [17]. The early inflammatory response, which often is associated with the blood vessels, starts immediately or a few hours after the onset of the ischemia and contributes to the irreversible damage [18-21]. Currently, there are two major ways used for treating ischemic stroke: (i) Dissolution of the clot in the occluded artery by a thrombolytic drug, rt-PA (recombinant tissue-plasminogen activator) [22] and, (ii) administration of neuroprotective agents [23]. Treatment with rt-PA is limited by time and should be administered within 4.5 hours after the onset of stroke to reduce the risk of hemorrhagic transformation [24, 25]. Moreover, rt-PA is associated with the risk of disruption to the blood-brain barrier (BBB) which is due to activation of matrix metalloproteinases [26]. Despite intense research, the results obtained with neuroprotective drugs in clinical trials have not revealed positive results [27, 28]. Hemorrhagic stroke Hemorrhagic stroke (15% of all strokes) is often associated with hypertension, and is due to the rupture of an arterial aneurysm or a vascular malformation [1, 29]. Hemorrhagic stroke is divided into two categories: intracerebral and subarachnoid hemorrhage. Intracerebral hemorrhage (ICH) is due to the rupture of a small artery (arterioles) which bleeds within the brain tissue. It is often associated with chronic high blood pressure and the symptoms often begin with severe headache. Subarachnoid hemorrhage (SAH) occurs when an artery or an arterial aneurysm on the surface of the brain ruptures and bleeds into the space between the pia mater and the arachnoid (subarachnoid space) [1]. The most common cause of the SAH is the spontaneous rupture of an arterial aneurysm. This is associated with acute rise of the intracranial pressure (ICP), reduction of cerebral blood flow (CBF), rapid discharge of blood into the basal cisterns, and delayed cerebral ischemia (DCI), each of which may be fatal. The SAH is most common in women and younger people (below 55 years old). Around 50-70% of patients with SAH die or suffer severe disability, and is the cause of up to 10% of all strokes [30-33]. The disease is biphasic, with an early/short-lived phase that occurs immediately after SAH with a reduction in CBF, followed by a chronic or prolonged phase which is characterized by a varying degree of pathological contraction of cerebral arteries, known as vasospasm [34, 35]. The vasospasm (narrowing of arteries) typically occurs within 5-15 days after SAH and is present in approximately one-third of patients and is accompanied by DCI [36, 37]. It can occur not only at the site of the hemorrhage, but also in brain arteries at a distance from the bleeding. The narrowing of the cerebral vessel lumen leads to reduction in local blood flow and in cerebral metabolism, causing severe cerebral ischemia, with increase in mortality of 1.5-3 folds during the first two weeks after SAH [37-39]. Despite intense research, the pathogenesis of DCI after SAH is not well understood and no specific pharmacological treatment is available. Current treatment recommendations involve management in an intensive care unit. The blood pressure is maintained with consideration to the patient’s neurologic status. In addition, calcium channel blockers, endothelin-1 receptor antagonists, hemodynamic management and endovascular treatment are also used, but these treatments are expensive, time-consuming and only partly effective [40]. Many theories have been advanced to explain the mechanisms responsible for vasospasm and DCI that occur after SAH such as, endothelial damage [41-43], enhanced smooth muscle cell (SMC) contractility, morphologic changes in vessel walls [44], enhanced levels of free radicals [45-47], increased production and release of potent vasomotor substances such as endothelin-1 (ET-1) and angiotensin II (Ang II) [48, 49], local inflammation and immunological reactions in the vascular wall [50-52]. Yet, the exact mechanisms underlying the vasospasm and the DCI remain unknown [53]. There is evidence that the amount of blood in the subarachnoid space is related to development of vasospasm [54]. Oxyhemoglobin from extravasated blood may be an important trigger of vasospasm and DCI after SAH [55-57] by inducing inflammation [50, 58]. It may in addition correlate with structural damage to the vessel wall [59], release of spasmogenic substances, and inhibition of endothelium dependent relaxation [60, 61]. It is suggested that the extravasated blood could induce generation of free radicals that subsequently may exert a direct local toxic effect on the cerebral arteries [62, 63]. G-protein coupled receptors following stroke Recently, a novel aspect of the pathophysiology of stroke has been suggested, namely that the upregulation of vasoconstrictor receptors in the cerebral arteries after stroke may be an important mechanism in the development of the final damage [64]. Vasoconstrictor receptors such as those of angiotensin II receptor type 1 (AT1) and endothelin-1 receptor type B (ETB) belong to the seven transmembrane G-protein coupled receptor (GPCR) family [65-67]. They are upregulated in the SMCs of cerebral vessels within and associated with the ischemic region after focal ischemic stroke [68] and after SAH [69]. This results in enhanced contractility of the vessels, which further impairs local blood flow and aggravates tissue damage. Importantly, the receptor ligands (angiotensin II and endothelin-1) are formed in the cerebrovascular endothelium. In addition, contractile responses mediated by AT1 and ETB receptors were found to be increased in SMCs of human cerebral arteries after organ culture [70]. Experimental stroke induces upregulation of cerebrovascular contractile receptors in the SMCs which are caused by increased receptor gene transcription induced via activation of specific intracellular signaling pathways (such as MEK-ERK1/2 and PKC pathways) [64]. Importantly, inhibition of these signaling pathways prevents the receptor upregulation, reduces infarct volume after ischemic stroke and improves neurological score and CBF after SAH [71, 72]. This may indicate that the increased cerebrovascular contractility caused by the upregulated receptors contributes to worsening of the brain damage. Inflammation in general and following stroke Inflammation is the body's defense against injurious factors and foreign antigens, e.g., trauma, infection and toxins, and is considered to be both a beneficial and a detrimental element of a pathological process. It is a complicated and multifaceted response, characterized by acute and chronic phases [73, 74]. Among many mechanisms involved in the pathogenesis of stroke, inflammation is increasingly recognized as a key factor. However, all the mediators of the inflammatory response have not been clearly identified [6, 75-77]. There is evidence to suggest that inflammation and immune responses are involved in all three stages of the ischemic cascade, from the acute intravascular process triggered by the interruption of the blood supply to the parenchymal processes that lead to brain damage and subsequently to tissue repair. The early inflammatory response contributes to the ischemic injury, whereas late responses may represent endogenous mechanisms of recovery and repair [78] (Figure 1). When there is a switch from detrimental to beneficial effects might depend on the strength and the duration of the stroke and knowledge about the mechanisms involved is crucial for determining the time-window for effective pharmacotherapy [79]. As mentioned above, reduction in CBF after stroke can result in energy depletion and subsequent neuronal cell death. This triggers an immune response that results in activation of a variety of inflammatory cells and molecules [51, 80, 81]. In the acute phase (minutes to hours), extravasated blood following SAH (or following reperfusion after arterial occlusion in transient ischemia) induces generation of reactive oxygen species (ROS). They may stimulate ischemic cells to produce inflammatory molecules such as cytokines and chemokines which in turn may activate microglial cells and increase leukocyte infiltration. These produce more cytokines, causing an increase in adhesion molecules, which are normally required for the adherence and accumulation of leukocytes and neutrophils to vascular endothelial cells and infiltration of brain parenchyma. In the sub-acute phase (hours to days), increased activation of inflammatory cells results in further production of pro-inflammatory mediators including more cytokines, extracellular MMPs, as well as iNOS which generates nitric oxide (NO) and more ROS [79, 82]. The intravascular accumulation of leukocytes and of platelets results in occlusion of microvessels, leading to hypoxia and further increases in levels of ROS [83, 84]. Activation of mast cells and macrophages can in turn lead to release of histamine (a strong vasoactive substance) and production of more cytokines and proteases [85]. In addition, degradation of extracellular matrix components by MMPs (mostly MMP-9) leads to BBB disruption which contributes to secondary brain damage by releasing serum and blood elements into the brain tissue resulting in vasogenic brain edema and post-ischemic inflammation [83]. Disruption and permeability of the BBB can be either transient or permanent depending on severity of the insult. Permanent disruption is associated with endothelial swelling, astrocyte detachment and blood vessel rupture in the ischemic area, while transient BBB disruption is caused by endothelial hyperpermeability to macromolecules in the penumbra area. This follows a biphasic pattern with an initial opening 2-3 hours after the onset of the insult and a second opening 24-48 hours after reperfusion leading to edema and increased intracranial pressure. All these events involve pro-inflammatory cytokines, adhesion molecules and production of MMPs [86, 87]. Cerebral blood vessels are the first to be exposed to the ischemic insults and their reaction to injury sets the stage for the inflammatory response. Post-ischemic inflammation thus involves activation of microglial and endothelial cells accompanied by migration of peripheral circulating inflammatory cells into the brain such as leukocytes, neutrophils, platelet, mast cells and macrophages. These events amplify signaling along inflammatory cascades increasing the accumulation of toxic molecules that enhance the secondary damage leading to more cell stress, edema, hemorrhage and finally cell death (Figure 1) [76, 79, 84]. On the other hand, many pro-inflammatory mediators play a positive role in late stage of stroke. For example, MMPs have been reported to promote brain regeneration and neurovascular remodeling in the later repair phase [79, 88, 89]. Moreover, macrophages and microglial cells also contribute to tissue recovery by scavenging necrotic debris, by producing anti-inflammatory cytokines and by facilitating plasticity [90] (Figure 1). Yet, despite these beneficial effects there is evidence that administration of anti-inflammatory drugs may reduce infarct volume and improved outcomes in animal models of stroke [91]. On the other hand, to date, clinical trials with anti-inflammatory agents have not been able to demonstrate improved clinical outcome [92, 93]. With better knowledge about which cells and molecules that participate and which mechanisms regulate the inflammatory reactions triggered by cerebral ischemia, it may be possible to identify novel targets for suppression of inflammation following cerebral ischemia and thereby develop more effective stroke therapies. Figure 1. Main inflammatory pathways that respond to injury after a stroke. The generation of ROS and free radicals that occur after stroke triggers inflammatory responses. This involves activation of cytokines and chemokines which leads to activation of inflammatory cells such as microglia and leukocytes causing more production of inflammatory mediators (cytokines, iNOS, MMPs and more ROS) which then lead to brain edema, hemorrhage and cell death. Thus, these early inflammatory responses contribute to ischemic injury, whereas late responses may represent endogenous mechanisms of recovery and repair through activation of anti-inflammatory cytokines, scavenging necrotic debris by microglia and neurovascular remodeling by MMPs. Major inflammatory mediators in cerebral ischemia In this present thesis, I have studied the expression of some of the major cytokines (IL-1ß, IL-6, TNF-α, TNF-R1 and R2), of MMP-9 (BBB associated) and of iNOS (potential toxic molecule) in cerebral vessel walls. Increased levels and activation of these factors may lead to exacerbation of vasoconstriction, resulting in decreased CBF and enhanced neuronal damage following a stroke. Cytokines Cytokines are recognized as small proteins, generally associated with inflammation, immune activation, cell differentiation and hematopoiesis [94]. Most cytokines are pleiotropic and have multiple biologic activities that generally act over a short distance, during short periods of time and at low concentrations. They are produced and expressed by different cell types such as astrocytes, macrophages, monocytes, microglia, platelets, endothelial and smooth muscle cells, neurons, fibroblasts and neutrophils [52, 95, 96]. Normally, they have a beneficial role, but when their expression increases in an imbalanced fashion they become detrimental [97]. Evidence for the involvement of cytokines in the pathology following stroke comes from the detection of their high levels in CSF and plasma of patients [98, 99]. It is thought that increased production and activation of such cytokines in vessel walls after cerebral ischemia/reperfusion may facilitate and expand the ischemic core by inducing secondary brain damage (brain swelling, impaired microcirculation, hemorrhage and inflammation) that typically develops after a delay of hours or days after the original ischemia, trauma or SAH [100]. It is well known that cytokines are involved in the upregulation and activation of adhesion molecules, MMPs, leukocytes, microglial, increased leukocyte-endothelium interaction and increase in vasoconstrictor substances like ET-1 following cerebral ischemia [52, 76, 101]. Tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) are the main cytokines which initiate inflammatory reactions and induce expression of other cytokines and inflammatory mediators after stroke. Ischemic brain has been shown to produce increased levels of TNF-α, IL-6 and IL-1ß, which are considered as a part of the damaging response [102]. Inhibiting the expression of these pro-inflammatory cytokines has been reported to reduce brain infarct size in animal models of stroke [103]. TNF-α TNF-α is a pleiotropic cytokine and exists as either a transmembrane or soluble protein. It is involved in the disruption of the BBB, as well as in inflammatory, thrombogenic and vascular changes associated with brain injury [104]. This cytokine promotes inflammation by stimulation of acute-phase protein secretion, enhances the permeability of endothelial cells to leukocytes, and the expression of adhesion molecules and other cytokines into the ischemic area [105, 106]. In addition, it has been suggested to stimulate angiogenesis after cerebral ischemia through induced expression of angiogenesis-related genes [107, 108]. It is known as a strong immunomediator, which is rapidly upregulated early in neuronal cells in and around the ischemic penumbra, and is associated with neuronal necrosis or apoptosis [105]. TNF-α effects are mediated via two receptors, TNF-R1 and TNF-R2, on the cell surface [109]. TNF-R1 is expressed on all cell types, can be activated by both membrane-bound and soluble forms of TNF-α and is a major signaling receptor for TNF-α. The TNF-R2 is expressed primarily on endothelial cells, responds to the membrane-bound form of TNF-α, and mediates limited biological responses [109]. There is evidence that TNF-α and its receptors may activate nuclear factor-κB (NF-κB), a transcription factor whose activation leads to expression of several genes involved in inflammation and cell proliferation [110-112]. In addition, NF-κB is involved in signaling cell death as well as cell survival, and the balance between these signals determines the toxic degree of TNF-α [112, 113]. TNF-α appears then to be not only neurotoxic but also neuroprotective. Increased TNF-α levels have been observed in brain tissue, plasma and CSF in several CNS diseases such as Alzheimer’s, multiple sclerosis and Parkinson’s [114-116]. Accordingly, a recent study demonstrated that blocking TNF-α significantly reduced infarct size after both permanent and transient MCAO, suggesting the involvement of TNF-α in neuronal cell damage [104]. In contrast, there is evidence to suggest that brain injury after ischemia becomes worse in mice lacking TNF-R1, suggesting that TNF-α mediates neuroprotection through this receptor [117]. The function of TNF-α appears to differ between brain regions. TNF-α released for instance in the striatum is considered as neurodegenerative, while release in the hippocampus has been suggested to promote neuroprotection [112]. Several investigators have suggested that the detrimental effects are activated in the early phase of the inflammatory

Cerebral ischemia induces microvascular pro-inflammatory cytokine expression via the MEK/ERK pathway

<p>Abstract</p> <p>Background</p> <p>Cerebral ischemia from middle cerebral artery wall (MCA) occlusion results in increased expression of cerebrovascular endothelin and angiotensin receptors and activation of the mitogen-activated protein kinase (MAPK) pathway, as well as reduced local cerebral blood flow and increased levels of pro-inflammatory mediators in the infarct region. In this study, we hypothesised that inhibition of the cerebrovascular inflammatory reaction with a specific MEK1/2 inhibitor (U0126) to block transcription or a combined receptor blockade would reduce infarct size and improve neurological score.</p> <p>Methods</p> <p>Rats were subjected to a 2-hours middle cerebral artery occlusion (MCAO) followed by reperfusion for 48 hours. Two groups of treated animals were studied; (i) one group received intraperitoneal administration of a specific MEK1/2 inhibitor (U0126) starting at 0, 6, or 12 hours after the occlusion, and (ii) a second group received two specific receptor antagonists (a combination of the angiotensin AT₁receptor inhibitor Candesartan and the endothelin ET_Areceptor antagonist ZD1611), given immediately after occlusion. The middle cerebral arteries, microvessels and brain tissue were harvested; and the expressions of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and phosphorylated ERK1/2, p38 and JNK were analysed using immunohistochemistry.</p> <p>Results</p> <p>We observed an infarct volume of 25 ± 2% of total brain volume, and reduced neurological function 2 days after MCAO followed by 48 hours of recirculation. Immunohistochemistry revealed enhanced expression of TNF-α, IL-1ß, IL-6 and iNOS, as well as elevated levels of phosphorylated ERK1/2 in smooth muscle cells of ischemic MCA and in associated intracerebral microvessels. U0126, given intraperitoneal at zero or 6 hours after the ischemic event, but not at 12 hours, reduced the infarct volume (11.7 ± 2% and 15 ± 3%, respectively), normalized pERK1/2, and prevented elevation of the expressions of TNF-α IL-1ß, IL-6 and iNOS. Combined inhibition of angiotensin AT₁and endothelin ET_Areceptors decreased the volume of brain damaged (12.3 ± 3; <it>P </it>< 0.05) but only slightly reduced MCAO-induced enhanced expression of iNOS and cytokines</p> <p>Conclusion</p> <p>The present study shows elevated microvascular expression of TNF-α, IL-1ß, IL-6 and iNOS following focal ischemia, and shows that this expression is transcriptionally regulated via the MEK/ERK pathway.</p

Enhanced cerebrovascular expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 via the MEK/ERK pathway during cerebral ischemia in the rat.

BACKGROUND: Cerebral ischemia is usually characterized by a reduction in local blood flow and metabolism and by disruption of the blood-brain barrier in the infarct region. The formation of oedema and opening of the blood-brain barrier in stroke is associated with enhanced expression of metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). RESULTS: Here, we found an infarct volume of 24.8 +/- 2% and a reduced neurological function after two hours of middle cerebral artery occlusion (MCAO), followed by 48 hours of recirculation in rat. Immunocytochemistry and confocal microscopy revealed enhanced expression of MMP-9, TIMP-1, and phosphorylated ERK1/2 in the smooth muscle cells of the ischemic MCA and associated intracerebral microvessels. The specific MEK1/2 inhibitor U0126, given intraperitoneal zero or 6 hours after the ischemic event, reduced the infarct volume significantly (11.8 +/- 2% and 14.6 +/- 3%, respectively; P < 0.05), improved neurological function, normalized expression of phosphorylated ERK1/2, and reduced expression of MMP-9 and TIMP-1 in the vessel walls. Administration of U0126 12 hours after MCAO did not alter the expression of MMP-9. Immunocytochemistry showed no overlap in expression between MMP-9/TIMP-1 and the astrocyte/glial cell marker GFAP in the vessel walls. CONCLUSION: These data are the first to show that the elevated vascular expression of MMP-9 and TIMP-1, associated with breakdown of the blood-brain barrier following focal ischemia, are transcriptionally regulated via the MEK/ERK pathway

Application of Lattice Boltzmann Method for Simulating Stably Stratified Flows past Cylinders

This research looks into the intriguing subject of ambient density-stratified flows, which have long captivated researchers due to their representation of real-world physical phenomena such as diapycnal mixing in oceans driven by environmental influences. The study specifically focuses on the flow past a cylindrical object within such stratified flows, which introduces complexities involving buoyancy and viscous effects. A major focus of this research is the examination of the lattice Boltzmann method as a novel approach to model stratified flows around circular cylinders by solving coupled Navier-Stokes and advection-diffusion equations. The study investigates the impact of stratification on wake characteristics and various flow parameters for a single cylinder at six Reynolds numbers ranging from 10 to 600 and Froude numbers from 2.19 to 7.51. Additionally, the investigation includes the case of two cylinders arranged in tandem at a Reynolds number of 100, with similar Froude numbers. This research demonstrates the suitability and robustness of the lattice Boltzmann method in modeling stratified flows past cylinders. The findings reveal that even moderate levels of stratification can significantly influence the wake pattern, potentially leading to changes in the flow regime. Moreover, the study demonstrates that the introduction of stratification is associated with a reduction in the drag coefficient and shedding frequency, leading to altered flow behaviors. Furthermore, in the case of flow past two cylinders, the presence of stratification increases the critical spacing between the cylinders

Position referenced force augmentation in teleoperated hydraulic manipulators operating under delayed and lossy networks: a pilot study.

Position error between motions of the master and slave end-effectors is inevitable as it originates from hard-to-avoid imperfections in controller design and model uncertainty. Moreover, when a slave manipulator is controlled through a delayed and lossy communication channel, the error between the desired motion originating from the master device and the actual movement of the slave manipulator end-effector is further exacerbated. This paper introduces a force feedback scheme to alleviate this problem by simply guiding the operator to slow down the haptic device motion and, in turn, allows the slave manipulator to follow the desired trajectory closely. Using this scheme, the master haptic device generates a force, which is proportional to the position error at the slave end-effector, and opposite to the operator's intended motion at the master site. Indeed, this force is a signal or cue to the operator for reducing the hand speed when position error, due to delayed and lossy network, appears at the slave site. Effectiveness of the proposed scheme is validated by performing experiments on a hydraulic telemanipulator setup developed for performing live-line maintenance. Experiments are conducted when the system operates under both dedicated and wireless networks. Results show that the scheme performs well in reducing the position error between the haptic device and the slave end-effector. Specifically, by utilizing the proposed force, the mean position error, for the case presented here, reduces by at least 92% as compared to the condition without the proposed force augmentation scheme. The scheme is easy to implement, as the only required on-line measurement is the angular displacement of the slave manipulator joints

The role of tumor necrosis factor-α and TNF-α receptors in cerebral arteries following cerebral ischemia in rat

<p>Abstract</p> <p>Background</p> <p>Tumour necrosis factor-α (TNF-α) is a pleiotropic pro-inflammatory cytokine, which is rapidly upregulated in the brain after injury. TNF-α acts by binding to its receptors, TNF-R1 (p55) and TNF-R2 (p75), on the cell surface. The aim of this study was first to investigate if there is altered expression of TNF-α and TNF-α receptors in cerebral artery walls following global or focal ischemia, and after organ culture. Secondly, we asked if the expression was regulated via activation of the MEK-ERK1/2 pathway.</p> <p>Methods</p> <p>The hypothesis was tested <it>in vivo </it>after subarachnoid hemorrhage (SAH) and middle cerebral artery occlusion (MCAO), and <it>in vitro </it>by organ culture of isolated cerebral arteries. The localization and amount of TNF-α, TNF-α receptor 1 and 2 proteins were analysed by immunohistochemistry and western blot after 24 and 48 h of organ culture and at 48 h following SAH or MCAO. In addition, cerebral arteries were incubated for 24 or 48 h in the absence or presence of a B-Raf inhibitor (SB386023-b), a MEK- inhibitor (U0126) or an NF-κB inhibitor (IMD-0354), and protein expression evaluated.</p> <p>Results</p> <p>Immunohistochemistry revealed enhanced expression of TNF-α, TNF-R1 and TNF-R2 in the walls of cerebral arteries at 48 h after MCAO and SAH compared with control. Co-localization studies showed that TNF-α, TNF-R1 and TNF-R2 were primarily localized to the cell membrane and the cytoplasm of the smooth muscle cells (SMC). There was, in addition, some expression of TNF-R2 in the endothelial cells. Immunohistochemistry and western blot analysis showed that these proteins were upregulated after 24 and 48 h in culture, and this upregulation reached an apparent maximum at 48 h of organ culture. Treatment with U0126 significantly reduced the enhanced SMC expression of TNF-α, TNF-R1 and TNF-R2 immunoreactivities after 24 and 48 h of organ culture. The Raf and NF-κB inhibitors significantly reduced organ culture induced TNF-α expression while they had minor effects on the TNF-α receptors.</p> <p>Conclusion</p> <p>The present study shows that cerebral ischemia and organ culture induce expression of TNF-α and its receptors in the walls of cerebral arteries and that upregulation is transcriptionally regulated via the MEK/ERK pathway.</p

Effect of Duration and Frequency of Acoustic Doppler Velocimetry Measurement on Calculation of Turbulent Flow Characteristics

Acoustic Doppler velocimetry (ADV) is one of the most suitable devices for measuring flow characteristics. Determination of measurement frequency and duration, in a way that the results are calculated with the lowest error, is very important. The goal of this study was to determine the optimum measurement frequency and duration to save money and time. 3D instantaneous subcritical flocharacteristicsts are measured at 200, 100, 25, and 5Hz frequencies for a duration of 3 minutes, in a laboratory flume with an aspect ratio of less than 5. Then, 3D averaged velocities, shear velocity, turbulence intensity, and Reynolds shear stress are calculated. Results show that the reduction of error is independent of the number of measured data and its dependence is on the data collection duration and frequency. For measurements of 3D averaged velocity components, the appropriate measurement frequency and duration are 1Hz and 50 seconds, respectively. To determine the shear velocity, using logarithmic law, reducing the frequency and duration, results in a maximum error of 13%. For calculation of turbulence flow characteristics, like turbulence intensity and Reynolds shear stress, the measurement frequency, and duration of up to 25Hz and 50-70sec, respectively, results in an error of less than 10%

In Vitro Antiplasmodial Activity and Cytotoxic Effect of (Z)-2-Benzylidene-4, 6-Dimethoxybenzofuran-3 (2H)-One Derivatives

Background: Aurones are naturally occurring compounds that belong to flavenoids family and have antiplasmodial effects. This study investigated some new aurones derivatives against chloroquine sensitive Plasmodium falciparum. Here we report the synthesis, in vitro antiplasmodial activity and cytotoxic evaluation of 11 compound from derivatives of (Z)-2- benzylidene-4, 6-dimethoxybenzofuran-3(2H)- one. Methods: The cytotoxic evaluations of active compounds were performed with MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium bromide) assay on human breast cancer cell lines; MCF7 and T47D. Results: From 11 compounds M3, M6 and M7 compounds showed good antiplasmodial effect against chloroquine-sensitive 3D strain of P. falciparum with IC50 (50% inhibitory concentration) values of 7.82, 7.27 and 2.3 µM respectively. No noticeable toxicity was observed with these compounds when tested against tested cell lines. Conclusion: The replacement of the 4 and 5 positions at ring B of aurone derivatives, with propoxy and bromide (Br) respectively was revealed highly advantageous for their antiplasmodial effect